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doi: 10.1242/10.1242/dev.00324

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Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation

¹ Genetic Disease Research Branch, National Human Genome Research Institute, National Institute of Health, Bethesda, MD 20892, USA
² Department of Medicine (School of Medicine), University of Pennsylvania, Philadelphia, PA 19104, USA

View larger version (116K): [in a new window]   Fig. 1. Wnt5a expression and delayed cell differentiation in the developing cartilage of the Wnt5a mutant. Sections of developing long bones were stained according to the Weigert-Safranin staining procedure. Proliferative chondrocytes are red. Gene expression was detected by in situ hybridization with 35S-labeled radioactive riboprobes on consecutive sections of the developing humerus at 13.5 dpc. (A) Hypertrophic chondrocytes (indicated by arrows) were not detected until 18.5 dpc in the radius (R) and ulna (U) region of the mutant (right). Hu, humerus. (B) Upper panel: osteoblast and blood vessel invasion (arrow) was incomplete in the mutant humerus. The hypertrophic region (double headed arrows) in the mutant (right) was longer than that in the wild-type (left) embryo, although the mutant humerus was much shorter overall. Lower panel: the boxed bone collar regions are shown at higher magnification. The bone matrix indicated by the arrows was reduced in the mutant. P, proliferative chondrocytes; H, hypertrophic chondrocytes. (C) Wnt5a was expressed at the boundary of proliferative and prehypertrophic chondrocytes where Ihh and Cbfa1 were expressed. Wnt5a expression flanked the expression domain of Col10a1. In the perichondrium, Wnt5a was expressed in the outer layer and Cbfa1 was expressed in the inner layer.

View larger version (81K): [in a new window]   Fig. 2. Significantly reduced Ihh expression, delayed chondrocyte and osteoblast differentiation in the Wnt5a mutant. (A) Early chondrogenic defect in the limb was examined using whole-mount in situ hybridization. In the Wnt5a mutant (below), Sox9 expression was not detected from the earliest stages of chondrogenic condensation in the distalmost limb mesenchyme (arrowheads). Ihh expression was also significantly reduced. The arrowhead indicates the future humerus. (B) Sections of forelimbs from 12.5 dpc embryos, except those hybridized with the Pthlh probe were from 13.5 dpc forelimbs. The expression of Wnt5a, Ihh and Col10a1 was reduced to below detectable levels in the cartilage of the mutant. Wnt5a expression in the distal limb (arrows) of the mutant was normal. Pthrpr and Cbfa1 expression was significantly reduced, but Pthlh (PTHrP in figure) expression (arrowheads) in the mutant was comparable with that in the wild-type littermate. (C) Strong Ihh expression was detected in the humerus and a few cells in the ulna in the mutant at 15.5 dpc (arrowheads); Ptch was also expressed in the ulna (arrowhead); Pthrpr expression (arrowhead) was weaker and no Col10a1 expression was detected; Cbfa1 expression was reduced in both chondrocytes and perichondrium (arrowhead); osteocalcin was not detected in the mutant. H, humerus; R, radius; U, ulna.

View larger version (72K): [in a new window]   Fig. 3. Reduced chondrocyte proliferation in the Wnt5a mutant. (A) BrdU labeled cells on sections of the distal humerus of 14.5 dpc embryos are shown. The chondrocyte region is divided into two zones before hypertrophy. Zone I is adjacent to the joint and composed of round cells. Zone II is composed of mostly flattened and columnar chondrocytes adjacent to the prehypertrophic chondrocytes. In the mutant (right), because there are no regular columnar chondrocytes, Zone II is half the region between the Zone I on each end of the cartilage. Zone II was shorter and contained fewer BrdU-labeled cells in the mutant, especially in the central region. The diameter of the cartilage was reduced in Zone I but expanded in Zone II when compared with the wild type. (B) Quantification of BrdU labeling experiments shown in A. Cell proliferation rate was higher in Zone II than in Zone I in the wild-type embryo. In the mutant, proliferation in Zone I was slightly increased, but proliferation in Zone II was reduced by about 30% in the mutant to a level similar to that in Zone I.

View larger version (63K): [in a new window]   Fig. 4. Overexpression of Ihh in the Wnt5a mutant. Skeletal preparations and sections (bottom row) of the forelimb of 18.5 dpc mouse embryos are shown. Ihh was overexpressed in the chondrocytes in the Col2a1-Gal4;UAS-Ihh mice. Joint formation was suppressed and ectopic ossification was observed in the elbow and knee regions (arrows). In the Col2a1-Gal4;UAS-Ihh; Wnt5a-/- mouse, the long bones were slightly longer than the Wnt5a-/- mutant (arrowheads), but much shorter than the Col2a1-Gal4;UAS-Ihh mutant. Suppression of joint formation and ectopic ossification in Col2a1-Gal4;UAS-Ihh; Wnt5a-/- embryos (arrows) resembled the Col2a1-Gal4;UAS-Ihh mutant, but in a much smaller region. H, humerus; R, radius; U, ulna; Fe, femur; T, tibia; Fi, fibula.

View larger version (80K): [in a new window]   Fig. 5. Overexpression of Wnt5a in chondrocytes delays chondrocyte differentiation. (A) Morphological analysis of Col2a1-Wnt5a embryos. H, humerus; R, radius; U, ulna. Upper panel: skeletal preparations of mouse embryos at 18.5 dpc. The Col2a1-Wnt5a mouse (right) had shorter snout, smaller rib cage, shorter limb long bones and reduced ossification (arrow) than did the wild type (left). Lower panel: sections of the forelimb at 15.5 dpc. Chondrocyte hypertrophy (arrow) is only detected in a small area in the humerus in the Col2a1-Wnt5a embryo. (B) Expression of markers for prehypertrophic chondrocytes (Ihh and Pthrpr) and hypertrophic chondrocytes (Col10a1) in 14.5 dpc embryos. Wnt5a is expressed at a higher level in Col2a1-Wnt5a number 25 than in Col2a1-Wnt5a number 17. In Col2a1-Wnt5a number 17, there is only one Ihh and Pthrpr expression domain and the Col10a1 domain is much smaller. The undifferentiated chondrocyte region in the wild-type embryo (black line) is shorter than that in Col2a1-Wnt5a number 17 (yellow line), but the entire skeletal element is longer in the wild-type embryo. In Col2a1-Wnt5a number 25, the expression of Ihh, Pthrpr and Col10a1 is not detected. (C) BrdU-labeled cells on sections of the distal radius of 14.5 dpc embryos are shown. Fewer chondrocytes were labeled by BrdU in the Col2a1-Wnt5a embryos, as Wnt5a was expressed at a higher level. The columnar chondrocyte structure was disrupted in the Col2a1-Wnt5a embryo.

View larger version (65K): [in a new window]   Fig. 6. Overexpression of Wnt5b delays chondrocyte differentiation. (A) Consecutive sections of the humerus at 15.5 dpc. Wnt5b was expressed in the prehypertrophic chondrocytes where Ihh was expressed. Its expression also extended into the hypertrophic region. Wnt5b expressing chondrocytes were more differentiated than Wnt5a-expressing chondrocytes. (B) Skeletal preparation of 17.5 dpc embryos. The Col2a1-Wnt5b embryo had an open skull (arrow), significantly shortened long bones and reduced ossification in the limb, and smaller rib cages. Forelimbs are shown separately. (C) Expression of markers for chondrocyte differentiation at 15.5 dpc. The expression of Wnt5a, Ihh and Col10a1 (ColX in figure) was not detected in the chondrocytes in the transgenic embryos. Expression in the digits was not affected (arrow). (D) BrdU labeled cells on sections of the distal humerus of 17.5 dpc embryos are shown. More chondrocytes were labeled by BrdU in the Col2a1-Wnt5b embryos, especially in Zone I. The columnar chondrocyte structure was disrupted in the Col2a1-Wnt5b embryo.

View larger version (130K): [in a new window]   Fig. 7. Distinct expression patterns of cyclin D1 and p130 in different chondrocyte zones and their regulation by Wnt5a and Wnt5b. (A) In the developing cartilage of a wild-type embryo at 18.5 dpc, p130 was expressed at a higher level in Zone I (double headed arrows) and differentiated chondrocytes (arrow), but cyclin D1 was expressed at a higher level in Zone II chondrocytes. In the Wnt5a-/- cartilage, cyclin D1 expression was decreased and Zone I was reduced. When Ihh was overexpressed in chondrocytes in the Col2a1-Gal4; Uas-Ihh embryo, cyclin D1 expression was increased in Zone II but p130 expression in Zone I was not affected. When Ihh was overexpressed in the Wnt5a-/-; Col2a1-Gal4; Uas-Ihh mouse, cyclin D1 expression was elevated and Zone I region was increased when compared with the Wnt5a-/- mutant mouse. (B) Cyclin D1 expression was reduced by Wnt5a overexpression in the Col2a1-Wnt5a embryo (above, 18.5 dpc), but increased by Wnt5b overexpression in the Col2a1-Wnt5b embryo (below, 17.5 dpc). (C) The zone with higher p130 expression (double headed arrows) was expanded in the Col2a1-Wnt5a embryo (above, 18.5 dpc), but reduced in the Col2a1-Wnt5b embryo (below, 17.5 dpc).

View larger version (62K): [in a new window]   Fig. 8. Wnt5a and Wnt5b have opposite activities in regulating Sox9-controlled Col2a1 expression. (A) Sox9 protein was examined by immunohistochemistry. Sox9 protein level was decreased in both Wnt5a-/- and Col2a1-Wnt5a mice. No change was detected in Col2a1-Wnt5b mice. (B) The expression of Col2a1, a direct target of Sox9, was decreased in Col2a1-Wnt5a mice but increased in Wnt5a-/- and Col2a1-Wnt5b mice. (C) Col2a1 reporter activity was decreased by Wnt5a and increased by Wnt5b in primary chondrocytes in a dose-dependent manner. The histograms are presented as the mean±s.d. from three independent experiments. (D) Col2a1 reporter and mutant Col2a1 reporter that lacks Sox9-binding sites could not be activated by TCF1. But Wnt1 inhibited the Col2a1 reporter.

View larger version (39K): [in a new window]   Fig. 9. Proposed model for the action of Wnt5a and Wnt5b in regulating chondrocyte proliferation and differentiation. The morphology of a developing long bone is schematically shown on the left. Molecular interactions in regulating chondrocyte proliferation and differentiation are shown on the right. The major findings of this study are that Zone I and Zone II chondrocytes express p130 and cyclin D1 differentially, and that Wnt5a and Wnt5b regulate multiple transitional steps between different chondrocyte zones. Wnt5a expression is in purple. Wnt5b expression is in light blue. We show that Wnt5a promotes the expression of p130 but inhibits the expression of cyclin D1. Wnt5a may also downregulate Sox9 transcription activity. We propose these are the underlying mechanisms whereby Wnt5a inhibits the transition from Zone I to Zone II, but promotes the transition from Zone II to the prehypertrophic region. Conversely, Wnt5b enhances Sox9 transcription activity and cyclin D1 expression. This promotes Zone II formation but inhibits cell cycle withdrawal and chondrocyte hypertrophy. Thus, Wnt5a and Wnt5b act in parallel pathways to the Ihh and PTHrP negative feedback loop to coordinate chondrocyte proliferation and differentiation. Sox9 and cell cycle regulators may be the common targets.

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